Artificial yeast plasmid cloning vectors are inherited nonrandomly at mitosis, with most copies going to the mother cell, and few to the bud; evidently, DNA (or chromatin) associates nonrandomly with higher-order nuclear structures. In contrast, copies of the natural yeast plasmid 2- micron circle are partitioned evenly; this requires two plasmid proteins, REPl and REP2, and a specific target sequence, STB. These proteins also regulate transcription from several plasmid promoters, but the cis-acting sites required for this are unknown. The purpose of this project is to determine how REP partitioning works, and whether it is mediated by the same DNA-protein interactions that regulate transcription. The experimental approaches will include general and site-specific mutagenesis of the 2-micron circle to obtain mutants in partitioning; deletion analysis of the STB site and the promoters affected by REP partitioning; construction of very large linear and circular plasmids containing one or more STB sites, to assess the "reach" of the partitioning effect of such sites; the selection of host mutations in REP partitioning, to identify specific interactions of the plasmid proteins with nuclear structures; and overproduction of the REP proteins (and deletion mutants of REP1) for in vitro examination of their DNA- binding properties. The potential significance of the results of these studies is that they may suggest ways in which subnuclear localization of DNA sequences in eukaryotic cells affects their expression, a problem of fundamental importance; and that they may also reveal how specific structural interactions impede the free movement of DNA through the nucleus, which has implications for the mechanism o production of chromosomal nondisjunction, an important cause of birth defects. Additionally, certain outcomes of the "large DNA stabilization" could allow construction of high-copy minichromosome vectors, a technological advance.